EP3328858A1 - Novel 6,7-dihydropyrido[2,1-a]phthalazin-2-ones for the treatment and prophylaxis of hepatitis b virus infection - Google Patents

Abstract

The invention provides novel compounds having the general formula: wherein R¹ to R⁶ are as described herein, compositions including the compounds and methods of using the compounds.

Description

Novel 6.7-dihydropyrido[2. 1 -a]phthalazin-2-oncs for the treatment and prophylaxis of hepatitis

B virus infect ion

The present invent ion relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in part icular to HBsAg (HBV Surface antigen) inhibitors and HBV DNA production inhibitors useful for treating treating HBV infect ion.

FIELD OF THE INVENTION

The present invent ion relates to novel 6,7-dihydropyrido[2, l -a]phthalazin-2-ones having pharmaceut ical act ivity, their manufacture, pharmaceut ical composit ions containing them and their potential use as medicaments.

The present invent ion relates to com ounds of formula I

wherein R¹ to R⁶ are as described below, or to pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

The hepatitis B v irus (HBV) is an enveloped, partially double-stranded DNA virus. The compact 3.2 kb HBV genome consists of four overlapping open reading frames (ORF), which encode for the core, polymerase ( Pol), envelope and X-proteins. The Pol ORF is the longest and the envelope ORF is located within it, while the X and core ORFs overlap with the Pol ORF. The lifecycle of HBV has two main events: 1) generation of closed circular DNA (cccD A) from relaxed circular ( RC DNA), and 2 ) reverse transcription of pregenomic RNA (pgRNA) to produce RC DNA. Prior to the infect ion o f host cells, the HBV genome exists within, the v irion as RC DNA. It has been determined that HBV virions are able to gain entry into host cells by non-specifically binding to the negat ively charged proteoglycans present on the surface of human hepatocytes (Schulze, A., P. Gripon & S. Urban. Hepatology, 46, (2007), 1759-68) and via the specific binding of HBV surface ant igens ( HBsAg) to the hepatocyte sodium- taurocholate cotransporting polypeptide (NTCP) receptor (Yan, H. et al. J Virol, 87, (2013), 7977-91). Once the virion has entered the cell, the viral cores and the encapsidated RC DNA are transported by host factors, via a nuclear local izat ion signal, into the nucleus through the

Impp/Impa nuclear transport receptors. Inside the nucleus, host DNA repair enzymes convert the RC DNA into cccD A. cccDNA acts as the template for all viral mRNAs and as such, is responsible for HBV persistence in infected individuals. The transcripts produced from cccDNA arc grouped into two categories; Pregenomic RNA (pgRNA) and subgenomic RNA. Subgenomic transcripts encode for the three envelopes (L, M and S ) and X proteins, and pgRNA encodes for Prc-Corc, Core, and Pol proteins (Quasdorff, M. & U. Protzer. J Viral Hepat, 17, (2010), 527- 36). Inhibition of HBV gene expression or HBV RNA synthesis leads to the inhibit ion of HBV viral replicat ion and ant igens production (Mao, R. et al. PLoS Pathog, 9, (2013 ), e l 003494; Mao, R. et al. J Virol, 85, (201 1 ), 1048-57). For instance, IFN-a was shown to inhibit H BV replication and viral HBsAg production by decreasing the transcription of pgRNA and subgenomic RNA from the H B V covalcnt ly closed circular DNA (cccDNA ) min ichromosome. ( Belloni, L. et al. J Clin Invest, 1 22, (201 2). 529-37; Mao, R. et al. J Virol, 85, (201 1 ), 1048-57). All H BV viral mRNAs are capped and polyadenylated, and then exported to the cytoplasm for translation. In the cytoplasm, the assembly of new v irons is init iated and nascent pgRNA is packaged with v iral Pol so that reverse transcription of pgRNA, via a single stranded DNA intermediate, into RC DNA can commence. The mature nucleocapsids containing RC DNA are enveloped with cellular l ipids and viral L, M, and S proteins and then the infect ious HBV particles are then released by budding at the intracellular membrane ( Locarnini, S. Semin Liver Dis, (2005 ), 25 Suppl 1 , 9- 1 9 ). Interestingly, non-infect ious particles arc also produced that greatly outnumber the infect ious virions. These empty, enveloped particles (L, M and S) are referred to as subviral particles.

Importantly, since subviral particles share the same envelope proteins and as infectious particles, it has been surmised that they act as decoys to the host immune system and have been used for H BV vaccines. The S, M, and L env elope proteins are expressed from a single ORF that contains three different start codons. All three proteins share a 226aa sequence, the S-domain, at their C- termini. M and L have addit ional pre-S domains, Pre-S2 and Pre-S 2 and Pre-S 1 , respect ively. However, it is the S-domain that has the HBsAg epitope ( Lambert, C. & R. Prange. Virol J, (2007). 4. 45 ).

The control of viral infect ion needs a tight surveillance of the host innate immune system which could respond within minutes to hours after infect ion to impact on the init ial growth of the v irus and limit the development of a chronic and persistent infect ion. Despite the available current treatments based on IFN and nucleos(t)ide analogues, the Hepatitis B virus (HBV) infection remains a major health problem worldwide which concerns an estimated 350 million chronic carriers who have a higher risk of liver cirrhosis and hepatocellular carcinoma.

The secretion of ant iviral cytokines in response to HBV infect ion by the hepatocytes and/or the intra-hepatic immune cells plays a central role in the viral clearance of infected liver. However, chronically infected patients only display a weak immune response due to v arious escape strategies adopted by the virus to counteract the host cell recognition systems and the subsequent antiviral responses.

Many observations showed that several HBV v iral proteins could counteract the init ial host cellular response by interfering with the viral recognition signaling system and subsequently the interferon (IFN) ant iviral activity. Among these, the excessive secretion of HBV empty subviral particles (SVPs, H BsAg) may participate to the maintenance of the immunological tolerant state observed in chronically infected patients (CHB). The persistent exposure to HBsAg and other viral antigens can lead to HBV-specific T-cell deletion or to progressive functional impairment (Kondo et al. Journal of Immunology (1993), 150, 4659-4671 ; Kondo et al. Journal of Medical Virology (2004), 74, 425 433; Fisicaro et al. Gastroenterology, (201 0), 138, 682-93;). Moreover HBsAg has been reported to suppress the function of immune cells such as monocytes, dendritic ceils (DCs) and natural killer (NK) cells by direct interaction (Op den Brouw et al. Immunology, (2009b), 1 26, 280-9; Woltman et al. PLoS One, (201 1 ), 6, e l 5324; Shi et al. J Viral Hepat.

(201 2 ), 19, C26-33; Kondo et al. ISRN Gasteroenterology, (201 3 ), Art icle ID 935295 ).

HBsAg quantificat ion is a significant biomarker for prognosis and treatment response in chronic hepatitis B. However the achievement of HBsAg loss and seroconversion is rarely observed in chronical ly infected patients but remains the ult imate goal of therapy. Current therapy such as Nucieos(t)ide analogues are molecules that inhibit HBV DNA synthesis but are not directed at reducing HBsAg level. Nucleos(t)ide analogs, even with prolonged therapy, hav e demonstrated rates of HBsAg clearance comparable to those observ ed naturally (between -1%- 2%) (Janssen et al. Lancet, (2005 ), 365, 123-9; Marcellin et al. N. Engl. J Med., (2004), 35 1 , 1 206- 1 7; Buster et al. Hepatology, (2007), 46, 388-94). Therefore, there is an unmet medical need to target H BsAg for HBV treatment (Wieland, S. F. & F. V. Chisari. J Virol, (2005 ), 79, 9369-80; Kumar et al. J Virol, (201 1 ), 85, 987-95; Woltman et al. PLoS One, (201 1 ), 6, e l 5324; Op den Brouw et al. Immunology, (2009b), 126, 280-9). SUMMARY QF THE INVENTION

Objects of the present invent ion are novel compounds of formula I, their manufacture, medicaments based on a compound in accordance with the invent ion and their production as well as the use of compounds of formula I as H BV inhibitors and for the treatment or prophylaxis of HBV infect ion. The compounds of formula I show superior anti-HBV act iv ity.

The present invent ion relates to a compound of formula I

wherein

R¹ , R\ R ' and R are independently selected from i <,alkyl, haloCi <,alkyl, hydrogen, halogen, amino, cyano, pyrrolidinyl, hydroxy, (Ί <,alkoxy, haloC .alkoxy. C\ -cycloalkylCVi.alkyloxy, phenylCi <,alkyloxy, hydroxyC ₁ <,alkyloxy, C ₁ <,alkoxyC ₁ <,alky loxy,

C| .alkylsulfonylC^' i <,alkyloxy. cyanoC ₁ <,alkyloxy, aminoCi ^alkyloxy, Ci <,alkylamino V

(.alkyloxy, d iC ₁ <,alkylaminoC ₁ <,alkyloxy, C ₁ <,a !kylca rbo n y 1 a m i no C ₁ <,a 1 k y lo x y , Ci_

(.alkylsulfonylaminoC i <,alkyloxy, C ₁ ,alkoxycarlx)nylam inoC ₁ <,alkyloxy, p yrazo 1 yl C ₁ <,a 1 k y lo x y , triazolylCi ^alkyloxy and monocyclic heterocycloalkylC ₁ .alkyloxy, wherein monocycl ic heterocyc!oalkyl is A^'-containing monocyclic heterocycloalkyk

R and R⁶ arc independently selected from hydrogen, (Ί ^alkyl, haloCi < alkyl or (' <.alkenyk or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

As used herein, the term "C u.alkyr alone or in combinat ion signifies a saturated, linear- or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, propyl, isopropyl, 1 -butyl, 2-butyl, tert-butyl and the like. Particular '^"(^*Ί oalkyP groups are methyl, ethyl, isopropyl and feri-butyl. Thc term "C. .alkenyl" denotes a monovalent linear or branched hydrocarbon group of 2 to 6 carbon atoms with at least one double bond. In particular embodiments, Ci <,alkenyl has 2 to 4 carbon atoms with at least one double bond. Examples of alkenyl include ethenyl, propenyl, prop-2-enyl, isopropenyl, n-butenyl, and iso-butenyl. Particular "Ci-ealkyl" group is prop-2-enyl.

The term "C ; -cycloalkyl^", alone or in combinat ion, refers to a saturated carbon ring containing from 3 to 7 carbon atoms, particularly from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohcptyl and the like. Particular "(\ ₇cycloalkyl" groups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term "C | .alko.xy^" alone or in combinat ion signifies a group (Ί <,alkyl-0-, wherein the "Ci ^alkyl^" is as defined above; for example methoxy, ethoxy, pro poxy, /.w-propoxy, «-butoxy, /w-butoxy, 2-butoxy, fer/-butoxy, pentoxy, hexyloxy, octyloxy and the like. Particular "G . ₆alkoxy" groups are methoxy, ethoxy and pro poxy.

The term "halogen" means fluorine, chlorine, bromine or iodine.

The term "haloCi <,alkyl" denotes a (Ί <,alkyl group wherein at least one of the hydrogen atoms of the (Ί <,alkyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of haloCi <,alkyl include monofluoro-. difluoro- or trifluoro-methyl, - ethyl or -propyl, for example 3,3, -t ri fl uo ro pro py 1 , 3,3- difluoropropyl, 2-fluoroethyl, 2.2- difluoro ethyl, 2,2,2-trifluoroethyl, fluoromethyl, difluoromethyl or trifiuoromethyl. Particular "haioCi_6alkyl" group is difluoromethyl or trifiuoromethyl.

The term "haloCi ^,alkoxy" denotes a (Ί .alkoxy group wherein at least one of the hydrogen atoms of the C|.₍,alkoxy group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of haloCi ^alkoxyl include monofluoro-, difluoro- or trifluoro-mcthoxy, -ethoxy or -pro oxy, for example fluoropro oxy, difluoropropoxy, trifluoropropoxy, fluoroethoxy. difluoroethoxy. trifluoroethoxy, fluoromethoxy,

difluoromethoxy or trifluoromethoxy. Particular "haloCi ^alkoxy^" group is 3-fluoropropoxy, 3,3 -difluoropropoxy, 3,3,3-trifluoropropoxy, 2-fluoroethoxy, 2,2-difiuoroethoxy, 2.2,2- trifluoroethoxy, fluoromethoxy, difluoromethoxy or trifluoromethoxy.

The term "amino" denotes a group of the formula -NR'R" wherein R' and R" are independently hydrogen, (Ί <,alkyl, i <,alkoxy, Cv-cycloalkyl, h c t e ro C ·. - c y c lo a 1 k y 1 , aryl or heteroaryl. Alternatively, R' and R", together with the nitrogen to w hich they are attached, can form a h e t e ro C ; - c y c loalkyl.

The term "carbonyl" alone or in combinat ion refers to the group -C(O)-.

The term "cyano" alone or in combinat ion refers to the group -CN. Thc term "Ci_₆alkylsulfanyl" denotes the group -S-R^". wherein R^" is a C^'u.alkyl group as defined above. Examples of C, <,alkylsulfanyl include methylsulfanyl and ethylsulfanyl.

The term "Ci.6alkylsulfonyl" denotes a group -SO₂-R', wherein R' is a C ,alkyl. group as defined above. Examples of (Y<,alkylsulfonyl include methylsulfonyl and ethylsulfonyl.

The term "monocyclic heterocycloalkyl^" is a monovalent saturated or partly unsaturated monocyclic ring system of 4 to 7 ring atoms, comprising 1 , 2, or 3 ring heteroatoms selected from . O and S, the remaining ring atoms being carbon. Examples for monocyclic

heterocycloalkyl are aziridinyl, o.xiranyl, azetidinyl, oxetanyl, pyrrol id in yl, 2 -o xo - pyrro 1 i d i n y 1 , tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, t et rahy d ro t h io p yra n y 1 , piperazinyl, morpholinyl, 2- oxo-morpholinyl, 2-oxo-piperazinyl, thio morpholinyl, 1 , 1 -dioxo-thiomorpholin-4-yl, 1 , 1 - dioxothiolanyl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Particular '^"monocyclic heterocycloalkyl" groups are oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, 1 . 1 -dioxothiolanyl, morpholinyl, 2-oxo-pyrrolidinyl, 2 -o xo - mo rpho I i n y 1 and 2-oxo-piperazinyl.

The term 'W-containing monocycl ic heterocycloalkyl^" refers to a monocycl ic

heterocycloalkyl wherein at least one of the heteroatoms is N. Examples for .V-containing monocyclic heterocycloalkyl are azetidinyl, pyrrol id inyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl. isoxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, morpholinyl, azepanyl, diazepanyl, homopiperazinyl or oxazepanyl . Particular ^".V-containing monocycl ic

heterocycloalkyl" groups are pyrrolidinyl, 2 -o.xo - pyrro lid inyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl, and more particularly tetrahydropyranyl and morpholinyl.

The term "cnant iomer" denotes two stereoisomers of a compound which are non- superimposablc mirror images of one another.

The term "diastereomer^" denotes a stereoisomer with two or more centers of chiral ity and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melt ing points, boil ing points, spectral properties, and reactiv it ies.

The compounds according to the present invent ion may exist in the form of their pharmaceut ically acceptable salts. The term "pharmaceut ically acceptable salt" refers to conventional acid-addit ion salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydro iodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as /7-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetra methyl ammonium hydroxide. The chemical modification of a pharmaceut ical compound into a salt is a technique well known to pharmaceut ical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowabil ity and solubility of compounds. It is for example described in Bast in R.J., et al., Organic Process Research &

Development 2000, 4. 427-435. Particular arc the sodium salts of the compounds of formula I .

Compounds of the general formula I which contain one or several chiral centers can either be present as racemates, diastereomeric mixtures, or optically act ive single isomers. The racemates can be separated according to known methods into the enant iomers. Particularly, diastereomeric salts which can be separated by crystallization are formed from the racemic mixtures by react ion with an optically active acid such as e.g. D- or [.-tartaric acid, mandelic acid, malic acid, lact ic acid or eamphorsulfonic acid.

INHIBITOR OF HBsAg

The present invent ion provides (i) a compound having the general formula I :

wherein

R ', R ^! and R⁴ are independent ly selected from C_halky!, haloCi <.alkyl, hydrogen, halogen, amino, cyano, pyrrol id iny I, hydroxy, (Ί <.alkoxy, haloCi <,alkoxy. ; -c y c lo a 1 k y 1 C 1 <<a I k y lo x y , phenylCi <,alkyloxy, hydroxyC i /.alkyloxy, C 1 , a 1 k o x yC 1 _<·, a 1 k y lo x y , 1 <,alkylsuHanylC 1 <,alkyloxy, Ci .a!kylsul fonylCi r.alkyloxy. cyanoC 1 <,alkyloxy, aminoC 1 <,alkyloxy, Ci .alkylaminoCi .

(.alkyloxy, d iC 1 .alkylaminoC 1 ^alkyloxy, 1 .alkylcarlxtnylam inoC 1 <,alkyloxy, Ci -

(, a 1 k y 1 s u 1 fo n y I a m i no C 1 .alkyloxy, pyrazo lylC 1 <,alkyloxy, triazolyl I .alkyloxy and monocyclic heterocycloalkylC 1 .alkyloxy, wherein monocyclic heterocycloalkyl is Λ^'-containing monocycl ic heterocycioalkyi; R⁵ and R⁶ are independently selected from hydrogen. C^' u.alkyl. haloC^'u.alkyl or C.v,alkenyl; or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

Another embodiment of present invention is (ii) a compound of formula I, wherein R¹ is hydrogen;

R³ is Ci <,alkoxy, haloCi ^alkoxy, hydroxyC i ^alkoxy, C i <,alkoxyC i <,alkoxy, Ci .

(.alkylsul fonylCi <,alkoxy, carboxyC | <,alkoxy, aminoCi .alkoxy, CV

(.alkoxycarbonylaminoC i .alkoxy, mo rpholi n y 1 C i <, a 1 ko x y or tetrahydropyranylC i <,alkoxy; R⁴ is hydrogen;

R⁵ is hydrogen;

R⁶ is hydrogen, or (V<,alkenyl;

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof. A further embodiment of present invention is (iii) a compound of formula I , wherein

R^! is hydrogen;

R² is methoxy;

R ^" methoxy, trifluoroethoxy, h yd ro x y d i m e t h y 1 p ro po x y , methoxypropoxy,

methy lsu 1 fo nyl propoxy. cyanopropoxy, carboxybutoxy, aminopentoxy. tert- bu t o x yea rbo n y 1 a m i no pe n t o x y , morpholinylethoxy or t e t ra h yd ro p yra n y 1 m e t ho x y ;

R⁴ is hydrogen;

R⁵ is hydrogen;

R⁶ is hydrogen, methyl, isopropyl, isobutyl or prop-2-enyl;

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof

Another embodiment of present invent ion is (iv) a compound of formula I or

pharmaceutically acceptable salts, or enant iomers, or diastereomer thereof, wherein R^" is methoxy, and all remain ing substituents hav e the significances giv en herein before. Another embodiment of present invent ion is (v) a compound of formula I or

pharmaceut ically acceptable salts, or enantiomers, or diastereomer thereof, w herein R³ is haloCV (.alkoxy, hydroxyC i <,alkoxy, C i <,alkoxy i <,alkoxy, cyanoCi .alkoxy, CV (,alkoxycarlx)nylaminoC i -(,alkoxy or tetrahydropyranylC ,alkoxy. and all remaining substituents have the significances given herein before.

A further embodiment of present invention is (vi) a compound of formula I or

pharmaceut ically acceptable salts, or enant iomers, or diastercomer thereof, wherein R ^" is trifluorocthoxy, h yd ro x yd i m cthylp ro p o x y , met ho x yp ro po x y , cya no pro poxy, t rt- butoxycarbonylaminopentoxy, or t et ra h yd ro p yra n y 1 m e t h o x y , and all remaining substituents have the significances given herein before. Another embodiment of present invent ion is (vii) a compound of formula I or

pharmaceut ically acceptable salts, or enant iomers, or diastercomer thereof, wherein R⁶ is Ci_ (,alkyl, and all remaining subst ituents have the significances given herein before.

A further embodiment of present invention is (viii) a compound of formula I or pharmaceut ically acceptable salts, or enant iomers, or diastercomer thereof, wherein R⁶ is isobutyl, and all remaining subst ituents have the significances given herein before.

Part icular compounds of formula I according to the invent ion are the following:

9, 1 0-Dimethoxy-2-oxo-6,7-dihydropyrido[2, 1 -a]phthalazine-3-carboxylic acid;

9, 1 0-Dimethoxy-6-methyl-2-oxo-7/7-pyrido[ 2, 1 -a]plrthalazine-3-carboxylic acid;

6-Allyl-9, 10-dimethoxy-2-oxo-7//-pyrido[ 2, 1 -a ]phthalazine-3-carlx xylic acid;

6-lsopropyl-9. 10-dimethoxy-2-oxo-7//-pyrido[ 2. 1 -a]phtluilazine-3-earboxylic acid;

6-lsobutyl-9. 1 0-dimethoxy-2-oxo-7//-pyrido[2. 1 -a]phthalazine-3-carlx)xyl ic acid;

6-!sobutyl- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-7/7-pyrido[2, 1 -a]phthalazinc-3- carboxylic acid;

6-Isobutyl-10-methoxy-2-oxo-9-(2,2,2-trifluoroethoxy)-7H-pyrido[2, l-a]phthalazine-3- carboxyl ic acid;

6-lsobutyl- 10-methoxy-2-oxo-9-(tetrahydropyran-4-yimethoxy)-7H-pyrido [2, 1 - a]phthalazine-3-carboxylic acid;

6-lsobutyl- 10-mcthoxy-9-(3-methylsulfonylpropoxy)-2-oxo-7/7-pyrido[ 2, 1 -a jphth 3-carboxylic acid;

6-Isobutyl- l 0-methoxy-9-(2-morpholinoethoxy)-2-oxo-7/7-pyTido[ 2. l -ajphthalazine-3- carl )xylic acid; 9-(3-Hydroxy-2,2-dimethyl-propoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l- a]phthalazine-3-carboxylic acid;

9-(4-Carboxybutoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l-a]phthalazine-3- carboxylic acid;

9-(3-Cyanopropoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l-a]phthalazine-3- carboxylic acid;

9-[ 5-( 7V/7-butoxycarbonylamino )pentoxy ]-6-i.sobutyl- 10-methoxy-2-oxo-7/7-pyrido[2, 1 - aJphtlialazinc-3-carboxylic acid;

9-(5-Aminopcntoxy)-6-iNobutyl- 10-methoxy-2^xo-7/7-pyrido[ 2J -a]phthalazinc-3- carboxylic acid hydrochloride

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

10. A compound according to claim 1 , selected from

6-Isobutyl- 1 0-methoxy-9-(3-methoxypro poxy )-2-oxo-7/7-pyrido[ 2. 1 -a]phthalazine-3- carboxylic acid ;

6-Isobutyl- 10-methoxy-2-oxo-9-(tetrahydropyran-4-yimethoxy)-7H-pyrido [2. 1 - a]phthalazine-3 -carboxylic acid ;

9-(3-Hydroxy-2,2-dimethyl-propoxy)-6-isobutyl- 1 0-methoxy-2-oxo-7//-pyrido[ 2, 1 - a]phthalazine-3 -carboxylic acid ;

9-[ 5-( 7V/7-butoxycarlx)tiylamino )pentoxy j-6-i.sobutyl- 1 0-methoxy-2-oxo-7/7-pyrido[ 2, 1 - a]phthalazine-3 -carboxylic acid ;

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

More particularly, the invention relates to the following compounds of formula I :

6-Isobutyl- 10-methoxy-9-(3-metluixypro poxy )-2-oxo-7//-pyrido[ 2, 1 -a jphthalazine-3- carboxylic acid ;

6-lsobutyl- 10-methoxy-2-oxo-9-(tetrahydropyran-4-yimethoxy)-7H-pyrido [ 2, 1 - a]phthaiazine-3 -carboxylic acid ;

9-(3-Hydroxy-2,2-dimethyl-propoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l- a]phthalazine-3 -carboxylic acid;

9-[ 5-( 7fcT/-butoxycarbonylamino )pentoxy]-6-i,sobutyl- 10-methoxy-2-oxo-7//-pyTido[2, 1 - a]phthaiazine-3 -carboxylic acid ;

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof. SYNTHESIS

The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R ¹ to R⁶ are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, react ion condit ions, abbreviat ions and symbols have the meanings well known to a person of ordinary skill in organic chemistry.

General synthet ic route for intermediates (Scheme 1)

Scheme 1

The intermediate of formula V can be prepared according to Scheme 1 . Compound II reacts w ith DMFDMA at a temperature betw een 0 and 80 °C to afford compound I I I, which reacts with ethyl formate in the presence of a base such as t-BuOK, and then the result ing reaction mixture is treated with an acid such as hydrochloric acid to afford compound IV.

Compound IV reacts with tert-b tyl Y-aminoearbamate in a suitable solvent such as EtOH at temperature between rt and 1 00 °C to give compound V.

General synthet ic route for Compounds I (Scheme 2)

Scheme 2

I

The compound of formula I can be prepared according to Scheme 2, wherein X is halogen; Y is halogen or triflate; Q ¹ is halogen, -0-S(0)₂CH₃ or -0-S(0)₂-(4-methylphenyi); R" is Ci_ (,alkyl. Reaction of substituted benzene VI with compound V in the presence of a base such as K >CO ; affords Compound VII, which undergoes intramolecular coupling reaction to give compound VIII. The coupling reaction can be carried out in the presence of a Pd catalyst such as PdBr₂ and a suitable base such as KOAc in a suitable solvent such as DMA at a temperature between room temperature and 1 50 °C. Compound VIII reacts with halide, mesylate or tosylate in the presence of a base such as K₂C0₃ in a solvent such as acetonitrile or DMF to give compound VI I I-l . Hydro iyzation of Compound VI 11-1. with an acid such as T FA or HQ in a suitable solvent such as DCM, or with a base such as lithium hydroxide in a suitable solvent such as EtOH/H₂0 or MeOH/H₂0 affords the compound of formula I. General synthetic route for Compounds 1-1 (Scheme 3)

Scheme 3

R⁸Q¹ ,

base,

4 solvent

The compound of formula 1-1 can be prepared according to Scheme 3, wherein Q¹ is halogen, -0-S(0)₂CH₃ or -0-S(0)₂-(4-methylphenyl); R⁸ is C.alkyl, haloC,„alkyl, (\

- c y c 1 o a I k y 1 C i <, a 1 k y 1 , phenylCi <,alkyl. hydroxyCi <,alkyl, Cu;alkoxyC ,alkyl, Ci .

(, a 1 k y 1 s u I fa n y ! C i <, alky], C i <,alky Isu IfonylC i <<alkyL cyanoCi ,<,alkyl, amino Ci <,alkyl, Ci_

<·, a I k y I a m i no C i <> a 1 k y I , d iC i <,alkylam inoC i <,alky 1, C i <,a I k y 1 c a rlx) n y I a m i no C i <,a Iky L Ci .

(.alkylsulfonylaminoC^'i <,alkyl, pyrazolylCi <,alkyl, triazolylC^'i - (.alkyl or monocyclic heterocycloalkylCi <,alkyl, w herein monocycl ic heterocycloalkyl is N- containing monocyclic heterocycloalkyl: R" is Ci <,alkyl. Debenzylat ion of Compound VI I 1-2 by hydrogenation is carried out in the presence of Pd/C in a solvent such as ethanol to afford Compound VIII-3. Then Compound VIII-3 reacts with halide, mesylate or tosylate in the presence of a base such as ₂CO₃ in a solvent such as DMF to give VIII-4. Hydrolyzation of Compound VI I I-4 with an acid such as TFA or HCl in a suitable solvent such as DCM. or with a base such as lithium hydroxide in a suitable solvent such as EtOH7H₂0 or MeOH/H₂0 affords the compound of formula I.

This invention also relates to a process for the preparation of a compound of formula I comprising (a) hydrolysis of a compound of formula (A) by using an acid or a base

(b) hydrolysis of a compound of formula (B) by using an acid or a base

wherein R ¹ to R⁶, R⁸ and R ^{1 1} are defined as above unless otherwise indicated.

In step (a) and (b), an acid is for example TFA or HQ; or a base is for example lithium hydroxide or sodium hydro ide.

A compound of formula I when manufactured according to the above process is also an object of the invent ion.

The invention also relates to a compound of formula I for use as therapeutically active substance.

Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invent ion and a therapeutical ly inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, compounds of formula (I) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate bu ffer, at pH 5. In another embodiment, the compounds of formula ( I ) are sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulat ion or as an aqueous solut ion. Compositions are formulated, dosed, and administered in a fashion consistent with good medical pract ice. Factors for considerat ion in this context include the particular disorder being treated, the particular mammal being treated, the clin ical condit ion of the individual pat ient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedul ing of admin istration, and other factors known to medical practitioners. The "effective amount" of the compound to be administered will be governed by such considerations, and is the min imum amount necessary to inhibit HBsAg. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.

In one example, the pharmaceut ically effect ive amount of the compound of the invent ion administered parenterally per dose will be in the range of about 0.01 to 100 mg/kg, alternat ively about 0.01 to 100 mg/kg of patient body w eight per day, with the typical in it ial range of compound used being 0.3 to 1 5 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, preferably contain from about 0. 1 to about 1000 mg of the compound of the invention.

The compounds of the invent ion may be administered by any suitable means, including oral, topical ( including buccal and subl ingual ), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administrat ion.

The compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such composit ions may contain components convent ional in pharmaceut ical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.

A typical formulat ion is prepared by mixing a compound of the present invent ion and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art. The formulat ions may also include one or more buffers, stabilizing agents, surfactants, w etting agents, lubricating agents, emulsifiers, suspending agents, preservat ives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known addit ives to provide an elegant presentation of the drug (i.e., a compound of the present invent ion or pharmaceut ical composit ion thereof) or aid in the manufacturing of the pharmaceut ical product (i.e., medicament ). An example of a suitable oral dosage form is a tablet containing about 0. 1 to 1 000 mg of the compound of the invent ion compounded with about 0 to 2000 mg anhydrous lactose, about 0 to 2000 mg sodium croscarmellose, about 0 to 2000 mg po 1 y v i n y 1 p yrro I i do n e (PVP) 30, and about 0 to 2000 mg magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solut ion of the PVP. The resulting composit ion can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound, for example 0. 1 to 1000 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solut ion may be filtered, e.g., using a 0.2 micron filter, to remove impurit ies and contaminants.

An embodiment, therefore, includes a pharmaceut ical composit ion comprising a compound of Formula I, or a stereoisomer o pharmaceutically acceptable salt thereof. In a further embodiment includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceut ically acceptable salt thereof, together with a pharmaceut ical ly acceptable carrier o excipient.

The following example A and B illustrate typical compositions of the present invention, but serve merely as representative thereof.

Example A

A compound of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composit ion:

Per tablet

Act ive ingredient 200 mg

M icrocrystalline cellulose 155 mg

Corn starch 25 mg

Talc 25 mg

Hydro ypropylmethylcellulo se 20 mg

425 mg

Example B A compound of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composit ion:

Per capsule

Act ive ingredient 1 00.0 mg

Corn starch 20.0 mg

Lactose 95.0 mg

Talc 4.5 mg

Magnesium stearate mg

220.0 mg

INDICATIONS AND METHODS OF TREATMENT

The compounds of the invention can inhibit HBsAg production, or secretion and inhibit H BV gene expression. Accordingly, the compounds of the invent ion arc useful for the treatment or prophylaxis of H BV infect ion.

The invention relates to the use of a compound of formula I for the inhibition of HBsAg production or secretion.

The invent ion relates to the use of a compound of formula I for the inhibit ion of H BV DNA production.

The invent ion relates to the use of a compound of formula I for the inhibit ion of H BV gene expression.

The invention relates to the use of a compound of formula I for the treatment or prophylaxis of H BV infection.

The use of a compound of formula I for the preparation of medicaments useful in the treatment or prophylaxis diseases that are related to HBV infection is an object of the invention.

The invention relates in particular to the use of a compound of formula I for the preparation of a medicament for the treatment or prophylaxis of HBV infection.

Another embodiment includes a method for the treatment or prophylaxis of HBV infection, which method comprises administering an effective amount of a compound of Formula I , a stereoisomer, tautomer, prodrug, conjugates or pharmaceutically acceptable salt thereof.

EXAMPLES

The inv ention will be more fully understood by reference to the follow ing examples. They should not, howev er, be construed as limit ing the scope of the invention. Abbreviatiotis used herein are as follows:

μ ΐ. : microliter

Li m : micrometer

μΜ: micro moles per liter

DMF: dimethyl formam ide

D MFD MA: Λ ^', ; V- d i m e t h y 1 fo r m a m i d e dimethyl acetal

DMA: ;V, Λ - D i met h y 1 a n i 1 i n e

DMSO-J6: deuterated dimethylsulfoxide

EtOAc: ethyl acetate

h or hr: hour

hrs: hours

IC₅₀: the half maximal inhibitory concentration

HPLC: high performance liquid chromatography l.C MS: Liquid chromatography/mass spectrometry

METHANOL-d₄: perdeuteromethano 1

M: molarity

mg: milligram

MHz: megahertz

min: minute

rriL: milliliter

mM: millimoles per liter

mm: millimeter

mmol: millimole

MS (ESI): mass spectroscopy (electron spray ionizat ion)

NMR: nuclear magnet ic resonance

N₂: nitrogen

rt: room temperature

Pd/C: palladium on activated carbon

prep-HPLC: preparative high performance l iquid chromatography

TFA: trifluoroacetic acid

δ: chemical shift

i-BuOK: potassium /erz-butylate GENERAL EXPERIMENTAL CONDITIONS

Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SPl system and the Quad 12/25 Cartridge module, ii) ISCO combi-flash chromatography instrument. Silica gel Brand and pore size: i) KP-SIL 60 A, particle size: 40-60 μιη; ii) CAS registry NO: Silica Gel: 63231 -67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.

Intermediates and final compounds were purified by preparative HPLC on reversed phase column using X Bridge™ Perp C_{i 8} (5 ,um, OBD™ 30 x 100 mm.) column or SunFire™ Perp Gig (5 iim, OBD™ 30 100 mm ) column.

LC/MS spectra were obtained using an A equity Ultra Performance LC - 3 1 00 Mass Detector or Acquity Ultra Performance LC - SQ Detector. Standard LC/MS conditions were as follows (running time 3 minutes):

Acidic condition: A: 0.1% formic acid in 3¾0; B: 0.1% formic acid in acetonitrile;

Basic condition: A: 0.05% NH LO in H₂0; B: acetonitrile:

Neutral condition: A: H O: B: acetonitrile.

Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H) \

The microwave assisted reactions were carried out in a Biotage Initiator Sixty or CEM Discover.

NM Spectra were obtained using Broker Avance 400MHz.

All reactions involving air-sensitive reagents were performed under an argon atmosphere. Reagents were used as received from, commercial suppliers without further purification unless otherwise noted.

PREPARATIVE EXAMPLES

Example 1: 9,10-Dimethoxy-2-oxo-6,7-dihydropyrido| 2,l-a| phthalazine-3-carboxyHc acid

Step 1: Preparation of tert-butyl 2-(dimethyIaminomethylene)-3-oxo-butanoate

To a stirred solution of tert-butyl 3-oxobutanoate (30.0 g, 0. 19 mol ) in 1 , 4-dioxane ( 500 ml) was added N..V- dimethyl fo r m a m i d e dimethyl acetal (113.0 g, 0.95 mol ). The mixture was stirred at 25 °C for 16 hrs, and then concentrated under reduced pressure. The residue was diluted with H₂0 (300 mL). The resulting mixture was extracted with EtOAc (200 ml. ) for three times. The combined organic layers were dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give crude tert-butyl 2-(dimethylaminomethyiene)-3-oxo-butanoate (40.0 g) as a dark yellow liquid, which was used directly in the next step without further purificat ion.

Step 2: Preparation of tert-butyl 4-oxopyran-3-earboxylate

To a stirred solution of tert-butyl 2-(dimethylaminomethylene)-3-oxo-butanoate (40.0 g, 0. 1 9 mol) and ethyl formate (27.8 g, 0.36 mol ) in THF (700 ml. ) was added -BuO (52.6 g, 0.47 mol ) portion wise at 0 °C. Then the mixture was allowed to warm to 25 °C and stirred at the same temperature for 16 hrs. Then the reaction was quenched by adding 1 M hydrochloric acid. The result ing mixture was extracted with EtOAc (200 ml .) for three times. The combined organic layers were washed with saturated aHCO ; aqueous solution and brine, dried over anhydrous Na₂S0₄, and concentrated under reduced pressure. The residue was purified by the flash column chromatography to give tert-butyl 4-oxopyran-3-carboxylate (9.5 g) as a dark yellow solid.

Step 3: Preparation of tert-butyl l -ethyI-4-oxo-pyridine-3-earbox late

A suspension of tert-butyl Λ - a m i no c a r b a m a t e (7.0 g, 53.0 mmol ) and tert-butyl 4- o o yra n - 3 - c a rbo x y late (6.9 g, 35.3 mmol ) in absolute EtOH ( 70 ml. ) was heated under reflux with st irring for 48 hrs. Then the mixture was cooled to rt and concentrated to give crude t rt- butyl l-ethyl-4-oxo-pyridine-3-carboxylate ( 1 .4 g) which was used in the next step without further purification.

Step 4: Preparation of l-bromo-2-(ehloromethyI)-4,5-dimethoxy-benzene

A mixture of (2-bromo-4,5-dimethoxy-phenyi)methanol ( 1 .0 g, 4.05 mmol) and SOCL (0.32 ml,, 4.46 mmol ) in DCM ( 10 ml. ) was st irred at rt for 3 hrs. The reaction mixture was then concentrated to give l-bromo-2-(chloromethyl)-4,5-dimethoxy-benzene (1.1 g) which was used in the next step without further purification.

Step 5: Preparation of tert-butyl 1 -| 2-{2-bromo-4,5-dimethoxy-plieiiyl)- 1 -methyl- ethyl|-4-oxo-pyridine-3-carbox late

A mixture of tert-butyl l -ethyl-4-oxo-pyridine-3-carboxylate ( 1 .4 g, 4.52 mmol), 1 -bromo- 2-(chloromethyl)-4,5-dimethoxy-benzene (2.4 g, 9.03 mmol ) and ₂CO₃ (1.3 g, 9.03 mmol ) in acetone (20 mL) was heated at 50 °C with stirring for 2 hrs. Then the mixture was cooled to rt and diluted with water (20 mL). The resulting mixture was extracted with. EtOAc (50 mL) for three times. The combined organic layers were washed with brine, dried over anhydrous Na_:.SO-i_: and concentrated under reduced pressure to give ttvt-butyl. l-[2-(2-bromo-4,5-dimethoxy- phenyl)-l-methyl-ethyl]-4-oxo-pyridine-3-carboxylate (2.6 g) which was used in the next step without further purification.

Step 6: Preparation of 2, l- a| phthalazinc-3-carboxyIate

A mixture of /cW-butyl. l-[2-(2-bromo-4,5-dimethoxy-phenyl)-l-methyl-ethyl]-4-oxo- pyridine-3-carboxylate (4.0 g, 7.42 mmol ), PdBr₂ (99 mg, 0.37 mmol ) and KOAc ( 1 .45 g, 14.84 mmol) in DMA ( 1 5 mL) was heated at 120 °C with stirring overnight. Then the mixture was cooled to rt and filtered. The filtrate was concentrated under reduced pressure and then diluted with water (30 mL). The mixture was extracted with EtOAc (30 mL) for three times. The combined organic layers were washed with brine, dried over anhydrous Na:SO.i, and

concentrated to give crude tert-butyl 9, 10-dimethoxy-2-oxo-6,7-dihydropyrido[2, l- a]phthalazine-3-carboxylate (2.0 g) which was used in the next step without further purification.

Step 7: Preparation of 9,10-dimethoxy-2-oxo-6,7-dihydropyndo|2,l-a| phthaiazine-3- carboxylic acid

To a solut ion of tert-butyl 9, 10-dimethoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthaiazine-3- carboxylate ( 1.00 mg, 0.28 mmol.) in a mixed solvent of THF (5 mL) and ILO (5 mL) was added LiOH^'H₂0 (1 18 mg, 2.8 mmol). The mixture was heated at 90 °C with stirring for 3 hrs. The mixture was cooled to rt and extracted with EtOAc (20 mL) for three times. The combined organic layers were washed with brine, dried over anhydrous and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 9, 10-dimethoxy-2-oxo-6,7- dihydropyrido[2, 1 -ajphthalazine-3-carboxyl ic acid ( 10 mg) as a light yellow solid, Ή NMR (400 MHz, DMSO-J6 ) δ ppm 3.87 (d, 6 H) 4.09 - 4.34 (m, 2 H), 7.05 (br. s., 1 H), 7. 1 3 - 7.33 (m, 1 11 ), 7.38 - 7.52 (m, 1 1 1 ), 7.58 (br. s., 1 I I ), 8.26 - 8.53 (m, 1 H); MS obsd. ( ESI ) [( M+ I I ) ] : 303. Example 2: 9,10-Dimethoxy-6-methyl-2-oxo-7//-pyrido |2,l-a| phthalazine-3- carboxylic add

A mixture of tert-butyl 9, 10-dimethoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthalazine-3- carboxylate (100 mg, 0.28 mmol), K₂C0₃ (77 mg, 0.56 mmol) and CH₃I (174 μί, 2.8 mmol) in. acetonitrile (10 mL) was heated at 90 °C with stirring for 3 hrs. The mixture was cooled down, to rt and diluted with water (10 ml. ). The resulting mixture was extracted with EtOAc (20 mL) for three times. The combined organic layers were washed with brine, dried over anhydrous Na₂S04, and concentrated to give crude feri-butyl 9. 10-dimethoxy-6-methyl-2-oxo-7//-pyrido[2, 1 - a]phthalazine-3-carboxyiate (95 mg) which was used in the next step without further purification.

Step 2% Preparation of 9,10-dimctlioxy-6-methyl-2-ovo-7//-pyndo|2, l -a|phthalazine- earboxylic add

A mixture of ferf-butyl 9, 10-dimethoxy-6-methyl-2-oxo-7//-pyrido[2. 1 -a jphthalazine-3- carboxylate (95 mg, 0.26 mmol) and CF3COOH (1 mL) in. DCM (10 mL) was stirred at rt for 3 hrs. The mixture was concentrated under reduced pressure and the residue was purified by prep- HPLC to give 9, 1 0-dimcthoxy-6-methyl-2-oxo-7//-pyrido[2, 1 -a]phthalazine-3-carboxy! ic acid ( 10 mg) as a light yellow solid, Ή NMR (400 MHz, DMSO-J,,) δ ppm 2.65 (s, 3 I I ) 3.81 - 3.94 (m, 7 H) 4.33 - 4.43 (m, 2 H) 7.00 - 7. 1 1 (m, 1 H) 7.50 - 7.57 (m, 1 H) 7.60 - 7.66 (m, 1 H) 8.57 (s, 1 H); MS obsd. ( ESI ) [(M+H ) ] : 317.

Example 3: 6-AIIyl-9,10-dimcthoxy-2-oxo-7//-pyrido|2,l -a| phtha!azine-3-carboxylic acid

To a solution of tert-butyl 9, 10-dimethoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthalazine-3- carboxylate ( 1 00 mg, 279 iimo! ) in DMF (3 m 1. ) was added ally! bromide (50.6 mg, 419 umol ) and potassium carbonate (77. 1 mg, 558 umol ). The result ing mixture was heated at 60 °C with stirring for 16 hrs, and then filtered. The filtrate was concentrated under reduced pressure to give tert-butyl 6-allyi-9, 10-dimethoxy-2-oxo-7H-pyrido[2, l-a]phthalazine-3-carboxyiate which was used in the next step without further purification.

Step 2: Preparation of 6-ally 1-9, 10-dimethoxy-2-oxo-7//-pyrido| 2, 1 -a| phthaIazine-3- earboxylic acid

To a solution of crude tert-butyl 6-allyl-9.10-dimcthoxy-2-oxo-7//-pyrido[2. 1 - a]phthalazine-3-carboxyiate ( 100 mg) in DCM (2 mL) was added 2,2,2-trifluoroacetic acid (65 μ [. ). The mi ture was st irred for 5 hours at rt, and then concentrated under reduced pressure. The residue was purified by prep-HPLC to give 6-allyl-9, 10-dimcthoxy-2-oxo-7//-pyrido[ 2, 1 - a]phthalazine-3-carboxylic acid (28 mg). Ή NMR (400 Hz, DMSO) δ ppm 8.46 (s, H I ), 7.64 (s, 1H), 7.55 (s, 1H), 7.06 (s, 1H), 6.04 - 5.87 (m, 1H), 5. 1 7 (m, 1H), 5.03 (m, 1H), 4.53 - 4.36 (m, 2H), 3.91 (s, 3H), 3.86 (s, 31 1 ), 3.41 (d, 2H); MS obsd. (ESI ) [( M+H ) j : 343.

Example 4: 6-lsopropy -9, 10-dimethoxy-2-oxo-7//-pyrido|2,l-a|phthaIazine-3- carboxylic acid

Step 1: Preparation of tert-butyl 6-isopropyl-9,10-dimethoxy-2-oxo-7//-pyrido|2,l - a] phthalazine-3-earboxylate

To a solution of tert-butyl 9, 10-dimethoxy-2-oxo-6,7-dihydropyrido[2, 1 -a]phthalazine-3- carboxylate (60 mg, 1 67 iimol) in DMF (2 mL) was added potassium carbonate (46.3 mg, 335 iimol ) and diisopropyl sulfate (61 mg, 335 iimol ). The mixture was heated at 80 °C with st irring for 14 hours, and then filtered. The filtrate was concentrated under reduced pressure to give crude tert-butyl 6-isopropyl-9, 10-dimethoxy-2-oxo-7H-pyrido[2, l-a]phthalazine-3-carboxylate, which was used in the next step without further purification.

Step 2: Preparation of 6-isopropyl-9,10-dimethoxy-2-oxo-7//-pyrido|2,l - a| phthalazine-3-earboxylic acid

To a solution of crude tert-butyl 6-isopropyl-9. 1 0-dimctho.xy-2-o.xo-7//-pyrido[2. 1 - a]phthalazine-3-carboxylate (62 mg) in DCM (5 ml. ) was added TFA (62 il l.). The mixture was stirred for 3 hours at rt, and then concentrated under reduced pressure. The residue was purified by prep-HPLC to give 6-isopropyl-9,10-dimethoxy-2-oxo-7H-pyrido[2, l-a]phthalazine-3- carboxylic acid (4 mg), Ή NMR (400MHz, DMSO) δ ppm 8.16 (s, 1H), 7.81 (s, 1H), 7.73 (s, 1 11 ), 7.13 (s, 1H), 4.37 - 4.28 (m. 2H), 3.94 (s, 31 1 ), 3.89 (s, 311 ). 2.53 - 2.5 1 (m, 1 11 ), 1 .39 (d, 611 ); MS obsd. ( ESI ) [( M + H ) ] : 345. Example 5: 6-lsobutyl-9, 10-dimethoxy-2-oxo-7//-pyrido|2,l-a|phthalazine-3- carboxylic acid

Step 1 : Preparation of tert-butyl 9, 10-dimethoxy-6-(2-methylallyl)-2-oxo-7//- pyrido [2,1 -a] phthala/.ine-3-earbox late

To a solution of tert-butyl 9, 10-dimethoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthalazine-3- carboxylate ( 1 00 mg, 279 iimo!) in DMF (3 ml. ) was added 3-bromo-2-mcthylprop- 1 -ene (75.3 mg. 558 umol ) and potassium carbonate (77. 1 mg, 558 iimol). The mixture was heated at 60 °C with stirring for 1 6 hours, and then filtered. The filtrate was concentrated under reduced pressure to give crude tert-butyl 9.1 ()-dimetho.xy-6-(2-methylal lyl)-2-oxo-7//-pyrido[2. 1 -a]phthalazine-3- carboxylate ( 130 mg) which was used in the next step without further purificat ion.

Step 2: Preparation of 9,10-dimethoxy-6-(2-methy!aIlyI)-2-oxo-7//-pyrido|2,l - a| phthalazine-3-carboxylic acid

To a .solution of crude feri-butyl 9, 10-dimethoxy-6-(2-methylallyl)-2-oxo-7//-pyrido[2, 1 - a]phthaiazine-3-carboxyiate ( 130 mg, 268 iimol ) in DCM (2 ml. ) was added TFA (99.2 il l. ). The mixture was stirred at rt for 5 hours, and then concentrated under reduced pressure. The residue was purified by column chromatography to give 9, 10-dimethoxy-6-(2-methylallyl)-2-oxo-7H- pyrido[ 2, 1 -ajphtha!azine-3-carboxylic acid (30 mg).

Step 3: Preparation of 6-isobutyl-9J 0-dimethoxy-2-oxo-7//-pyrido|2, l-a|phthalazine- 3-carbox lic add

To a solution of 9, 10-dimethoxy-6-(2-methyiallyl)-2-oxo-7H-pyrido[2, l-a]phthalazine-3- carboxylic acid (30 mg, 84.2 iimol ) in EtOH (3 ml.) was added Pd/C (6 mg). The mixture was stirred under an at mosphere of hydrogen at rt for 16 hours. The mixture was filtered and the filt ate was concentrated under reduced pressure to give 6-isobutyl-9, 10-dimethoxy-2-oxo-6,7- dihydro-2/7-pyrido| 2. 1 -a]phthalazine-3-carlx)xy! ic acid ( 13 mg), Ή NM (400 MHz, DMSO) δ ppm 8.40 (s, 1H), 7.62 (s, 1 11 ), 7.55 (s, 1H), 7.09 (s, 1H), 4.42 (m, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 2.55 (d, 2H), 1 .69 - 1 .56 (m, 1H), 0.91 (d, 6H); MS obsd. ( ESI ) [( M+H )^' j: 359. Example 6: 6-lsobutyl-10-methoxy-9-(3-rnethoxypropoxy)-2-oxo-7//-pyrido|2,l - a| phthalazine-3-earboxylie add

Step 1 : Preparation of 5-benzyloxy-2-bromo-4-methoxy-benzaldehyde

To a solution of 2-bromo-5-hydroxy-4-methoxy-benzaidehyde (25 g, 108 mmol) in MeCN (500 ml . ) was added (chloromethyl )benzene ( 1 3.7 g, 108 mmol ) and potassium carbonate (29.9 g. 2 1 6 mmol ). The mixture was heated at 80 °C with stirring for 1 5 hours. After being cooled to rt, the mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was partitioned between brine and DCM. The organic layer was dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give 5-(benzyloxy)-2-bromo-4-methoxy- benzaldehyde (34.2 g, 96.9 mmol) which was used in the next step without further purificat ion.

Step2: Preparation of (5-benzylox -2-bromo-4-methoxy-phenyl)methanol

To a solution of 5-(benzyloxy)-2-bromo-4-methoxy-benzaldehyde (35 g, 106 mmol) in MeOH (600 ml .) was added aBH i (4 g, 1 06 mmol) portion-w ise at 0 °C. The reaction mixture was allow ed to warm to rt, st irred at rt for three hours, and then quenched with water. The mixture was concentrated under reduced pressure. The residue was partit ioned betw een brine and DCM. The organic layer was dried over anhydrous and concentrated under reduced pressure to give (5-(benz.yloxy)-2-bromo-4-methoxy-phenyl)methanol (32.5 g, 100 mmol), which was used in the next step without further purification.

Step3: Preparation of l -benzylox -4-bromo-5-(ehloromethyl)-2-methoxy-benzene

To a solution of (5-(benzyioxy)-2-bromo-4-methoxy-phenyi)methanoi (5 g, 14.7 mmol) in DCM (50 ml. ) was added SOCi₂ (1.61 ml., 22 mmol) drop-wise at rt. The mixture was stirred at rt for two hours, and then concentrated under reduced pressure to give l -(benzyloxy)-4-bromo-5- (chioromethyi)-2-methoxy-benzene (5 g, 13.8 mmol, 93.6 % yield ), which was used in the next step without further purificat ion.

Step4: Preparation of tert-butyl l -|(5-benzyloxy-2-bromo-4-methoxy-phenyl)methyI- ii'r^-butoxyearbonyl-amino|-4-oxo- ridine-3-earboxylate

To a solution of l -(benzyloxy)-4-bromo-5-(chloromethyl)-2-methoxy-benzene (5.0 g, 14.6 mmol) in MeCN ( 1 00 ml. ) was added potassium carbonate (4.05 g, 29.3 mmol ) and fert-butyi 1 - ((ter/-butoxycarbonyl)amino)-4-oxo-l ,4-dihydropyridine-3-carboxylate ( 14 g, 29.3 mmol ). The mixture was heated at 80 °C with stirring for 5 hours. After being cooled to rt. the mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to give fert-butyl l -[(5-benzyioxy-2-bromo-4-methoxy-phenyl)methyi- teri-butoxycarbonyl-amino]-4-oxo-pyridine-3-carboxylate (5.3 g, 7.75 mmol ).

StepS: Preparation of tert-butyl 9-benzyloxy- 10-methoxy-2-oxo-6,7- dihydropyrido|2J -a| phthalazine-3-carbox\ late and di-tert-butyl 9-benzyloxy- 10-methoxy- 2-oxo-7//-pyrido 2,l -a| phthalazine-3,6-diearbox late

To a 1 5 mL microwave v ial was added fert-butyl l -[(5-benzyloxy-2-bromo-4-methoxy- phenyl)methyi-teri-butoxycarbonyl-amino]-4-oxo-pyridine-3-carboxylate (700 mg, 1 . 14 mmol ), palladium (II) bromide (90.8 mg, 34 1 iimol), potassium acetate (223 mg, 2.27 mmol) and DMF (5 mL). The vial was capped and heated under microwave at 1 00 °C for 5 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure to give a mixture of tert- butyl 9-benzyloxy- 10-methoxy-2-oxo-6.7-dihydropyrido[2, 1 -a ]phthalazinc-3-carboxylate and di- fert-butyl 9-benzyloxy- 1 0-methoxy-2-oxo -7H-pyrido [2, 1 -a]phthaiazine-3 ,6-dicarboxylate, which was used in the next step without further purification.

Step 6: Preparation of 9-benzyloxy-10-methoxy-2-oxo-6,7-dihydropyrido|2,l - a| phthaIazine-3-earboxylic acid

To a solution of the crude mixture of tert-butyl 9-benzyloxy- 1 0-mcthoxy-2-oxo-6,7- dihydropyrido[2, l -a]phthalazine-3-carboxylate and di-tert-butyl 9-benzyloxy- 1 0-methoxy-2- oxo-7//-pyrido[ 2, l -a |phthaiazinc-3,6-dicarboxylate (4.0 g) in DCM (20 ml .) was added TFA (20 mL). The mixture was stirred at rt for 15 hours, and then concentrated under reduced pressure. Thc residue was treated with a mixture solvent of petroleum ether/ethyl acetate ( 1 0: 1 , V/V). The mixture was filtered and the filter cake was dried under reduced pressure to give 9-benzyloxy- 10-methoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthalazine-3-carboxylic acid (2.0 g).

Step7: Preparation of methyl 9-benzyIoxy-10-methoxy-2-oxo-6,7-dihydropyrido|2,l - a| phthalazine-3-earboxylate

To a suspension of 9-benzyloxy-10-methoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthalazine- 3-carboxyiic acid (0.70 g, 1.85 mmol ) in MeOH (20 mL) was added catalytic amount of concentrated sulfuric acid (50 il l . ). The mixture was heated at 80 °C for 20 hours. The mixture was concentrated under reduced pressure, and the residue was neutralized with saturated aqueous aHC^'O ;. The resulting mixture was extracted with DCM. The organic layer was dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give methyl 9-benzyloxy-10- methoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthalazine-3-carboxylate (703 mg, 1 .79 mmol ).

Step 8: Preparation of 9-benzyloxy-10-methoxy-6-(2-methylallyl)-2-oxo-7//- pyrido|2,l -a| phthalazine-3-earbox lic acid

To a solution of methyl 9-benzyloxy-10-methoxy-2-oxo-6,7-dihydropyrido[2, l- a]phthaiazine-3-carboxyiate (0.20 g, 510 iimol) in DMF (20 mL) was added sodium hydride (61.2 mg, 1 .53 mmol ) at 0 °C. The mixture was stirred for 30 minutes, and then 3-bromo-2- methylprop-l-ene (275 mg, 2.04 mmol) was added. The resulting mixture was allowed to warm to rt and stirred at rt for 40 hours. To the mixture was added water and sodium hydroxide (40.8 mg, 1 .02 mmol). The resulting mixture was stirred for additional two hours, and then acidified with 6 M hydrochloric acid, and extracted with DCM. The organic layer was dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give crude 9-benzyloxy-lO- methoxy-6-(2-methylailyl)-2-oxo-7H-pyrido[2, l-a]phthaiazine-3-carboxylic acid (241 mg). which was used in the next step without further purification.

Step 9: Preparation of 9-hydroxy-6-isobutyI- 10-methoxy-2-oxo-7//-pyrido|2,l- a| phthaIazine-3-earboxyIic acid

To a solution of crude 9-benzyloxy- 10-methoxy-6-(2-methylallyl )-2-oxo-7H-pyrido[2, 1 - a]phthalazine-3-carboxylic acid (24 1 mg) in EtOH (15 ml. ) was added Pd/C (50 mg). The mixture was stirred under hydrogen atmosphere at rt for 16 hours. Then the mixture was filtered and the filtrate was concentrated under reduced pressure to give crude 9-hydroxy-6-isobutyl- 1 0- methoxy-2-oxo-7//-pyrido[2, 1 -a jphthalazinc-3-carboxylic acid ( 1 23 mg), which was used in the next step without further purification.

Step 10: Preparation 6-isobutyl- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-7//- pyrido|2,l -a| phthalazine-3-carbox lic acid

To a solution of crude 9-hydroxy-6-isobutyl- 1 0-methoxy-2-oxo-7/7-pyrido[2. 1 - a]phthaiazine-3-carboxylic acid ( 60 mg) in DMF (3 ml. ) was added potassium carbonate (74.2 mg, 537 iimol) and 1 -bro nx) -3 -met hox y pro pane (61 .6 mg, 402 iimol). The mixture was heated at 100 °C with stirring for three hours, and then cooled to rt. To the mixture was added water (3 ml. ) and sodium hydroxide ( 10.7 mg, 268 iimol). The result ing mixture was stirred at rt for two hours, then acidified with 6 M hydrochloric acid and extracted with DCM. The organic layer was concentrated under reduced pressure, and the residue was purified by prep-I IPLC to give 6- isobutyi-10-methoxy-9-(3-methoxypropoxy)-2-oxo-7H-pyrido[2, l-a]phthaiazine-3-carboxyiic acid (17 mg). Ή NM (400MHz, DMSO-d₆) δ ppm 8.40 (s, H ), 7.62 (s, 1H), 7.55 (s, 1H), 7. 10 (s, 1 1 ), 4.46 - 4.36 (m, 2H), 4. 1 1 (t, 2H), 3.90 (s, 3H), 3.48 (t, 2H), 3.26 (s, 3H), 2.55 (d, 211 ), 2.05- 1 .95(m, 21 1 ), 1 .69 - 1 .57 (m. 1 11 ), 0.91 (d, 611 ); MS obsd. ( ESI ) [( M+H ) ^' ] : 41 7.

Example 7: 6-lsobutyl- 10-methoxy-2-oxo-9-(2,2,2-trifluoroethoxy)-7//-pyrido|2, l- a| phthalazine-3-earboxylic acid

To a solution of crude 9-hydroxy-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2,l- a]phthalazine-3-carboxylic acid (60 mg) in DMF (3 mL) was added potassium carbonate (74.2 mg, 537 iimol) and 1.1,1 -trifluoro-2-iodoet ane ( 169 mg, 805 iimol). The mixture was heated at 100 °C with stirring for 20 hours. After being cooled to rt. the reaction mixture was part itioned between 1 M hydrochloric acid and DCM. The organic layer was concentrated under reduced pressure, and the residue was purified by prep-HPLC to give 6-isobutyl- 10-metho.xy-2-o.xo-9- (2,2.2-trif1uoroetho.xy)-7//-pyrido[2, 1 acid (7 mg). Ή NMR

(400MHz, DM SO -J/,) ό ppm 8.42 (s, 1H), 7.72 (s, 111), 7.62 (s, 1 1), 7.22 (s, 1H), 4.90-4.78 (m, 211), 4.48 - 4.35 (m, 211), 3.94 (s, 311), 2.54 (d, , 211), 1.66-1.56 (m.111), 0.91 (d, 6H); MS obsd. (ESI ) [(Mill) ]: 427.

Example 8: 6-Isobutyl-10-mcthoxy-2-oxo-9-(tctrahydro|vran-4-ylmethoxy)-7//- pyrido|2,l-a|phthalazine-3-carbox lic acid

To a solution of crude 9-hydroxy-6-isobutyl- 10-methoxy-2-oxo-7//-pyrido[2, 1 - a]phthalazine-3-carboxylic acid (60 mg) in DMF (3 mL) was added potassium carbonate (74.2 mg, 537 iimol) and 4-(iodomethyl)tetrahydro-2//-pyran (91 mg, 402 mol). The mi ture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixture was added water (3 ml.) and sodium hydroxide (10.7 mg, 268 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified with 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 6-isobutyl- 10-metho xy-2-oxo-9-(tetrahydropyran-4- ylmethoxy)-7//-pyrido[2,l-a]phthalazine-3-carlxxylic acid (5 mg), Ή NMR (400 MHz, CDC1₃) δ ppm 8.60 (s, HI), 7.20 (s, 111), 7.04 (s, HI), 6.72 (s, HI), 4.36 - 4.16 (m, 211).4.08 - 4.01 (m. 2H), 3.94 (s, 3H), 3.92 (d.2H), 3.52 - 3.43 (m, 211).2.53 (d.2H).2.24 - 2.19 (m, 111).2.05 - 1.99 (m.111).1.69-1.60 (m, 211), 1.54 - 1.42 (m, 211), 0.96 (d, 611). MS obsd. (ESI ) [(M+H)^']: 443.

Example 9: 6-lsobulyl-10-metlioxy-9-(3-methyIsulfonylpropoxy)-2-oxo-7//- pyndo|2,l-a|phthalazine-3-carbox lic acid

To a solut ion of crude 9-hydroxy-6-isobutyl- 10-metho.xy-2-oxo-7//-pyrido[2, 1 - a]phthalazine-3-carboxylic acid (60 mg) in DMF (3 ml.) was added potassium carbonate (74.2 mg, 537 iimol) and 3 -( met hy lsu 1 fo ny 1 )propyl 4-methylbenzenesulfonate (118 mg, 402 iimol). The mixture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixture was added water (3 ml.) and sodium hydroxide (10.7 mg, 268 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified with 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 6-isobuty- 10-methoxy- 9-(3-methylsuifonyipropoxy)-2-oxo-7H-pyrido[2,l-a]phthalazine-3-carboxyiic acid (7.8 mg), ¹11 NMR (400MHz, DMSO-J,,) δ ppm 8.40 (s, 111), 7.64 (s, 111), 7.55 (s, 111), 7.09 (s, 111), 4.44 - 4.38 (m, 211), 4.22 - 4.15(m, 211), 3.91 (s, 311), 3.27 - 3.22 (m, 211), 3.04 (s, 311), 2.54 (d, 211). 2.23 - 2.16 (m, 211), 1.66 - 1.56 (m.111), 0.91 (d, 611); MS obsd. (ESI ) [(M+H)^']: 465. Example 10: 6-lsobutyl-10-methoxy-9-(2-morpholinoethoxy)-2-()xo-7//-pyndo|2,l- a|phtlialazine-3-carboxylic acid

To a solution of crude 9-hydroxy-6-isobutyl- 10-metho.xy-2-o.xo-7//-pyrido[2, 1 - a]phthaiazine-3-carboxylic acid (57 mg) in DMF (3 ml.) was added potassium carbonate (88.1 mg, 637 iimol) and 4 -( 2 - bro mo et h y 1 ) mo rp ho line hydrobromidc (87.6 mg, 319 umol) The mixture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixtu e was added water (3 ml.) and sodium hydroxide ( 10.2 mg, 255 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified with 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 6-isobutyl- 1 ()-methoxy-9-(2- morpholinoethoxy)-2-oxo-7//-pyrido[2.1 -a]phthalazine-3-carboxy1ic acid ( 10 mg), 'HNMR (400MHz, CDCis) δ ppm 8.60 (s, 1 1), 7.21 (s, 1H), 7.05 (s, 111), 6.75 (s, 111), 4.30-4.23 (m, 4H), 3.95 (s, 311), 3.76 - 3.73 (m, 411), 2.93-2.89 (m, 211), 2.65 - 2.62 (m, 411), 2.53 (d, 211), 1.68 - 1.61 (m.111), 0.97 (d, 611); MS obsd. (ESI ) [(M+H)^"]: 458.

Example 11 : 9-(3-Hydroxy-2,2-dimethyl-propoxy)-6-isobutyl-10-methoxy-2-oxo-7//- pyrido|2,l-a|phthalazine-3-earboxylic add

To a solution of crude 9-hydroxy-6-isobutyl- 10-methoxy-2-oxo-7//-pyrido[2, 1 - a]phthalazine-3-carboxylic acid (57mg, 127 iimol) in DMF (3 ml.) was added potassium carbonate (88.1 mg, 637 iimol) and 3-bromo-2,2-dimethylpropan-l-ol (85.2 mg, 510 iimol). The mixture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixture was added water (3 ml.) and sodium hydroxide (10.2 mg, 255 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified with 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 9-(3-hydroxy-2,2-dimethyl-propoxy)-

6-i.sobutyl- 10-methoxy-2-oxo-7//-pyrido[2, 1 -a]phtha!azine-3-carboxylic acid (7 mg), ¹11 MR (400MHz, DMSO-J6) δ ppm 8.39 (s, 1H), 7.61 (s, 111), 7.52 (s, 1H), 7.09 (s, 111), 4.67-4.61 (m, 111), 4.42-4.37 (m, 211), 3.90 (s, 311), 3.78 (s, 211), 3.30 - 3.28 (m, 211), 2.54 (d, 211), 1.65 - 1.58 (m.111), 0.95 (s, 611), 0.90 (d, 611); MS obsd. (ESI ) [(M+H)^']: 431.

Example 12: 9-(4-Carboxy butoxy)-6-isobuty 1- 10-methoxy-2-oxo-7//-py rido 12, 1 - a|phthaIa/.iiie-3-earboxylic acid

To a solution of crude 9-hydroxy-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2,l- a]phthaiazine-3-carboxylic acid (57mg, 127 iimol) in DMF ( 3m .) was added potassium carbonate (88.1 mg.637 iimol) and methyl 5-bromopentanoate (74.6 mg, 382 iimol). The mixture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixture was added water (3 mL) and sodium hydroxide (20.4 mg, 510 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified ith 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 9-(4-carboxybutoxy)-6-isobutyi-10- metho.xy-2-o.xo-7/7-pyrido[2, 1 -a]pluhalazine-3-carbo.xylic acid (7 mg), ¹ H NMR (400 Hz, DMSO-d₆) δ ppm 8.39 (s, 1H), 7.61 (s, IH), 7.53 (s, 11), 7.08 (s, 1H), 4.44-4.36 (m, 211), 4.09- 4.03 (m, 2H), 3.90 (s, 311), 2.53 (d, 211), 2.33 - 2.26 (m, 211), 1.81 - 1.73 (m, 211), 1.69 - 1.59 (m, 3H), 0.91 (d, 611). MS obsd. (ESI ) [(M+H) ]: 445.

Example 13: 9-(3-Cyanopropoxy)-6-is()butyl-10-iTicthoxy-2-oxo-7//-pyndo|2,l- a|phthalazine-3-earboxylic acid

To a solution of crude 9-hydro.xy-6-isobutyl- 10-metho.xy-2-o.\o-7//-pyrido[2, 1 - a]phthalazine-3-carboxylic acid (57 mg, 127 iimol) in DMF (3 ml.) was added potassium carbonate (88.1 mg, 637 umol) and 4 -b ro mo b u t a n c n i t r i 1 e (56.6 mg, 382 iimol). The mixture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixture was added water (3 mL) and sodium hydroxide ( 10.2 mg, 255 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified ith 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep-HPLC to give 9-(3-cyanopropoxy)-6-isobutyl-10-methoxy-2-oxo-

7H-pyrido[2,l-a]phthaiazine-3-carboxylic acid (10 mg), Ή NMR (400MHz, DMSO-</₆) δ ppm 8.40 (s, 1 1), 7.63 (s, 111), 7.55 (s, 111), 7.12 (s, IH), 4.45-4.36 (m.211), 4.16-4.09 (m, 211), 3.90 (s, 311), 2.70-2.62 (m, 211), 2.54 (d, 211), 2.09 - 2.05 (m, 211).1.66-1.57 (m.111), 0.91 (d, 611). MS obsd. (ESI ) [(M+H) ]: 412.

Example 14: 9-|5-(7^"i'ri-butoxycarbonylamino)penloxy|-6-isobutyl-10-methoxy-2-oxo- 7//-py rido 12,1-aj phthalazine-3-carbox He acid

To a solution of crude 9-hydroxy-6-isobutyl- 10-methoxy-2-oxo-7//-pyrido[2.1 - a]phthaiazine-3-carboxylic acid (57 mg, 127 umol) in DMF (3 ml.) was added potassium carbonate (88.1 mg, 637 iimol) and 5-((iert-butoxycarbonyl)amino)pentyl 4- methylbenzenesulfonate ( 137 mg, 382 iimol). The mixture was heated at 100 °C with stirring for 3 hrs, and then cooled to rt. To the above mixture was added water (3 ml.) and sodium hydroxide ( 10.2 mg, 255 iimol). The resulting mixture was stirred at rt for 2 hrs, and then acidified with 6 M hydrochloric acid. The mixture was partitioned between brine and DCM. The organic layer was separated and concentrated under reduced pressure. The residue was purified by prep -H PLC to give 9-[5-(teri-butoxycarbonyiamino)pentoxy]-6-isobutyi-10-methoxy-2-oxo-7H-pyrido[2,l- a]phthalazine-3-carboxyiic acid (13 mg), Ή NMR (400 MHz, DMSO-i¾) δ ppm 8.39 (s, 1H), 7.61 (s, III), 7.53 (s, 111), 7.08 (s, 1H), 6.83-6.77 (m, 1H), 4.43-4.36 (m, 211), 4.07-4.00 (m, 2H), 3.89 (s, 3H), 2.97 - 2.89 (m, 2H), 2.54 (d, 2H), 1.79 - 1.71 (m, 2H), 1.66-1.56 (m, 1H), 1.46 - 1.34 (m, 13H), 0.90 (d, 6H). MS obsd. (ESI ) [(M+H)⁺]: 530.

Example 15 : 9-(5-Aminopentoxy)-6-isobiityl- 10-methoxy-2-oxo-7//-pyrido [2,1- a|phthalaziiie-3-earboxylic acid hydrochloride

To a solution of 9-[5-(teri-butoxycarbonylamino)pentoxy]-6-isobutyl-10-methoxy-2-oxo-

7//-pyrido[2.1 -ajphthalazine-3-carlxxylic acid (8 mg, 15.1 iimol) in MeCN (0.5 ml) was added hydrochloric acid (5.51 iiL, 181 μπιοΐ). The mixture was stirred at rt for 2 hrs, and then concentrated under reduced pressure to 9-(5-aminopcntoxy)-6-i.sobutyl-10-methoxy-2-oxo-7//- pyrido[2,l-a]phthalazine-3-carboxylic acid hydrochloride (7 mg), ¹ H NMR (400 MHz, DMSO-J,) δ ppm 8.40 (s, 1H), 7.90-7.79 (m, 311), 7.61 (s, 111), 7.54 (s, 1H), 7.09 (s, 111), 4.45-4.36 (m, 211), 4.09-4.02 (m, 211), 3.90 (s, 311), 2.85 - 2.76 (m, 2H), 2.56 - 2.52 (m, 211), 1.83 - 1.77 (m, 211), 1.67 - 1.58 (m, 3H), 1.52 - 1.42 (m, 2H), 0.91 (d, 6H); MS obsd. (ESI ) [(M+H) ]: 430. BIOLOGICAL EXAMPLES

Example 16 materials and methods

HBV cell line

HepG2.2.15 cells (Acs et al. Proc Natl Acad Sci US A, 84, (1987), 464 1 -4 ), a

constitutively HBV-expressing cell line were cultured in DMEM+Glutamax-I medium

( Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bov ine serum ( Invitrogen) and G418 ( Invitrogen ) at a final concentration of 200 mg/L and maintained in 5% C0₂ at 37°C. HBsAg Assay

HepG2.2. 1 5 cells were seeded in duplicate into white, 96-well plates at 1.5 x 10⁴ cells/well. The cells were treated with a three-fold serial dilution series of the compounds in DMSO. The final DMSO concentration in all wells was 1% and DMSO was used as no drug control.

The HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou. China, Catalog number: CL03 1 0-2 ) was used to measure the levels of secreted HBV ant igens sem i-q uant itat i vcl y . For the detection 50 μ L well culture supernatant was used and HBsAg was quant ified using HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnost ics Co., Zhengzhou, China, Catalog number: CL03 1 0-2 ), 50 μ L of the supernatant was transferred to the CLIA assay plate and 50 μ L of enzyme conjugate reagent was added into each well. The plates were sealed and gently agitated for 1 hour at room temperature. The

supernatant-enzyme-mixture was discarded and wells were washed 6 times with 300 μ L of PBS. The residual l iquid was removed by plat ing the CL IA plate right side down on absorbent tissue paper. 25 μ L of substrates A and B were added to each ell. Luminance was measured using a luminometer (Mithras LB 940 Mult imode M icroplate Reader) after 1 0 minutes incubat ion. Dose- response curves were generated and the IC₅₀ value was extrapolated by using the E-WorkBook Suite ( ID Business Solutions Ltd., Guildford, UK). The IC₅₀ was defined as the compound concentration (or condit ioned media log dilution) at which HBsAg secret ion was reduced by 50% compared to the no drug control. The compounds according to formula I were tested for their capacity to inhibit HBsAg as described herein. The Examples were tested in the above assay and found to have IC₅₀ below 50 μΜ. Particular compounds of formula I were found to have IC₅₀ below 1 .0 μΜ. Results of HBsAg assay are given in Table 1 .

Table 1 : List of Activity data

Example No. IC50 (μΜ) Example No. IC50 (μΜ) Example No. IC50 (μΜ)

1 18.707 6 0.053 1 1 0.039

2 2.534 7 0. 1 72 1 2 9.428

3 0.74 8 0.044 1 3 0.1 1

4 26.068 9 0.624 14 0.047

5 0.248 10 0.225 1 5 0.5

Claims

Claims

1 . A compound of formula I,

wherein

R¹, R², R³ and R⁴ are independently selected from C ,alkyl, haloCi <,alkyl, hydrogen, halogen, amino, cyano, pyrrolidinyl, hydroxy, Ci <,alkoxy. haloCi <,alkoxy, C ; -c y c loalkyl C i <,a 1 k y lo x y , plienylCi <,alkyloxy, hydroxyC i _(>alkyloxy. C i << a 1 k o x yC _t ^,alky 1 o x y . C i ^alkyLsul fany!C i <,alky loxy. C i 1 k y 1 su I fo ny 1C i <>a 1 k y lo xy , cyanoC i <,a!kyloxy, aminoCi <,alkyloxy, Ci <,alkylaminoCi (.alkyloxy, d i C i <, a 1 k y 1 a m i n o C i ..<, a 1 k y lo x y , C i <,a 1 k y 1 c a rbo n y I a m i no C i <,a I k y lo x y , CY

a 1 k y 1 s u 1 fo n y I a m i no C i <,alkyloxy, C i <,alkoxycarlx nylam inoC i <>alkyloxy, pyrazo lylC i <,alkyloxy. 1 riazo 1 y 1 C i <,a Iky lo xy and monocyclic heterocycloalkylCi .alkyloxy, wherein monocyclic heterocycloalkyl is A-containing monocyclic heterocycloalkyl;

R⁵ and R⁶ are independently selected from hydrogen, C ,alkyl, haloCi .alkyl or CVr.aikenyl; or pharmaceutically acceptable salts, or enantiomers, or diastercomer thereof.

2. A compound of formula I according to claim 1 , wherein

R is hydrogen;

is Ci <_<alkoxy;

is Ci <,alkoxy, haloCi <,alkoxy, C i <,a!koxy i <>alkoxy, i

(.alkylsulfonylCi <,alkoxy, aminoCi .alkoxy, Ci_

(,alkoxycarlx)nylam inoC i <,alkoxy, mo r p ho 1 i n y 1 C i ..<, a 1 ko x y or t etrahydropyranylC i .alkoxy;

R⁴ is hydrogen;

R⁵ is hydrogen;

R⁶ is hydrogen, Ci <,alkyl or C^alkenyl:

or pharmaceutically acceptable salts, or enantiomers, or diastercomer thereof

3 A compound of formula I according to claim 1 or 2, wherein

R¹ is hydrogen;

R is methoxy; R³ methoxy, trifluoroethoxy, hydroxydimethylpropoxy, methoxypropoxy,

methyisulfonyipropoxy, cyanopropoxy, carboxybutoxy, aminopentoxy, tert- butoxycarbonylaminopentoxy, morphol in y lethoxy or tetrahydropyranylmethoxy;

R⁴ is hydrogen;

R⁵ is hydrogen;

R⁶ is hydrogen, methyl, isopropyl, isobutyl or prop-2-cnyl;

or pharmaceut ically acceptable salts, or enantiomers, or diastereomer thereof.

4. A compound of formula I according to any one of claims 1 to 3, or pharmaceutically acceptable salts, or enant iomers, or diastereomer thereof, wherein R is methoxy.

5. A compound of formula I according to any one of claims 1 to 4, or pharmaceut ically acceptable salts, or enant iomers, or diastereomer thereof, wherein R³ is haloCi <,alkoxy, h yd ro x y C i <, a 1 k o x y , C i <,a IkoxyC i <,alkoxy , cyanoC u.alkoxy, C i <,a I ko x y c a rbo n y 1 a m i no C i <,a 1 ko x y or t e t ra h yd ro p yra n y 1 C i <>alkoxy.

6. A compound of formula I according to any one of claims 1 to 5, or pharmaceut ically acceptable salts, or enant iomers, or diastereomer thereo wherein R³ is trifluoroethoxy, h yd ro x yd i m et h y 1 p ro po x y , methoxypropoxy, cyanopropoxy, tert-butoxycarbonyiaminopentoxy, or t et rah yd ro p yra n y 1 m et ho x y .

7. A compound of formula I according to any one of claims 1 to 6, or pharmaceut ically acceptable salts, or enantiomers, or diastereomer thereof, wherein R⁶ is Ci <,alkyl.

8. A compound of formula I according to any one of claims 1 to 7, or pharmaceut ically acceptable salts, or enant iomers, or diastereomer thereof, wherein R⁶ is isobutyl.

9 A compound according to claim 1 , selected from

9, 10-Dimethoxy-2-oxo-6,7-dihydropyrido[2, l-a]phthaiazine-3-carboxyiic acid;

9. 10-Dimethoxy-6-methyl-2-oxo-7//-pyrido|2, 1 -a]phthalazine-3-carboxylic acid;

6-Ailyl-9, 10-dimethoxy-2-oxo-7H-pyrido[2, l-a]phthaiazine-3-carboxylic acid;

6-lsopropyl-9, 10-dimethoxy-2-oxo-7//-p>Tido[ 2, 1 -a jphthalazinc-3-carboxyl ic acid;

6-Isobutyi-9, 10-dimethoxy-2-oxo-7H-pyrido[2, l-a]phthaiazine-3-carboxylic acid; 6-lsobutyl- 1 0-methoxy-9-(3-methoxypropoxy)-2-oxo-7H-pyrido[2. 1 -a]phthalazine-3- carboxylic acid;

6-Isobutyl-10-methoxy-2-oxo-9-(2,2,2-trifluoroethoxy)-7H-pyrido[2, l-a]phthalazine-3- carboxylic acid;

6-l.sobutyl- 10-methoxy-2-oxo-9-(tctrabydropyran-4-ylmcthoxy)-7//-pyrido [2, 1- a ] ph thalazi nc-3 -carlx) xy 1 i c acid;

6-Isobutyl-10-methoxy-9-(3-methylsulfonylpropoxy)-2-oxo-7H-pyrido[2, l-a]phthalazine- 3-carboxylic acid;

6-Isob tyl-10-methoxy-9-(2-mo holinoethoxy)-2-oxo-7H-pyrido 2, l-a]pllthalazine-3- carboxylic acid;

9-(3-Hydroxy-2,2-dimethyl-propoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l- a]phthalazine-3 -carboxylic acid;

9-(4-Carboxybutoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l-a]phtlialazine-3- carboxylic acid;

9-(3-Cyanopropoxy)-6-i.sobutyl- 1 0-methoxy-2-oxo-7/7-pyrido[2, 1 -a |phthalazine-3- carboxylic acid;

9-[ 5-( 7V/7-butoxycarlx)nylamino )pcntoxy ]-6-i.sobutyl- 10-mcthoxy-2-oxo-7//-pyrido[ 2, 1 - a ] pht ha 1 az i nc-3 -carboxy 1 i c acid;

9-(5-Aminopcntoxy)-6-isobutyl- 10-methoxy-2-oxo-7/7-pyrido[ 2, 1 -a]phthalazinc-3- carboxylic acid hydrochloride

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

10. A compound according to claim 1 , selected from

6-Isobutyl- 1 0-methoxy-9-(3-methoxypropoxy)-2-oxo-7/7-pyrido[2, 1 -a]phtha!azine-3- carboxylic acid ;

6-Isobutyi- 10-methoxy-2-oxo-9-(tetrahydropyran-4-ylmethoxy)-7/7-pyrido [ 2, 1 - a]phthalazine-3 -carboxylic acid ;

9-(3-Hydroxy-2,2-dimethyi-propoxy)-6-isobutyl-10-methoxy-2-oxo-7H-pyrido[2, l- a]phthaiazine-3 -carboxylic acid ;

9- [5 -(rert-butoxycarbonylamino)pentoxy] -6-isobutyi- 10-methoxy-2-oxo-7/7-pyrido [ 2, 1 - a]phthalazine-3 -carboxylic acid ;

or pharmaceutically acceptable salts, or enantiomers, or diastereomer thereof.

1 1 . A process for the preparation of a compound according to any one of claims 1 to 1 0 comprising

(a) hydrolysis of a compound of formula (A)

(b) hydrolysis of a compound of formula (B)

wherein R¹ to R⁶ are defined as in any one of claims 1 to 8, R⁸ is i <,alkyl, haloCi <,alkyl. CY -cycloalkylC i <,alkyl, phenyl i .alkyl, hydro.xyCi <,alkyl, (Ί .alkoxyCi <,alkyl, Ci_

(.alkylsulfany i <,alkyl, C i <,alky sulfonylC i <,alkyl, cyanoCu.alkyl, amino C_halky!, CY

(.alkylaminoC i <,alkyl, diCY^alkylaminoC i <,a!kyl, C i <, a 1 k y 1 c a rbo n y I a m i no C i <, a I k y 1 , CY

₍,a Iky lsu 1 fo nyla minoC i <,alkyl, C i <>a 1 ko x yea rbo n y 1 a m i no C i <,alkyl, pyrazolylC i ,alkyl, triazo lylC i _ (.alkyl or monocyclic h c t e ro c y c lo a I k y I i <, a I k y 1 , wherein monocycl ic heterocycloalkyl is N- containing monocycl ic heterocycloalkyl; R" is CY<,alkyl.

12. A compound according to any one of claims 1 to 10 for use as therapeutically active substance.

13. A pharmaceutical composit ion comprising a compound in accordance with any one of claims 1 to 10 and a therapeutically inert carrier.

14. The use of a compound according to any one of claims 1 to 10 for the treatment or prophylaxis of HBV infection.

1 5. The use of a compound according to any one of claims 1 to 1 0 for the preparation of a medicament for the treatment or prophylaxis of HBV infect ion.

16. The use of a compound according to any one of claims 1 to 1 0 for the inhibit ion of HBsAg production or secretion, or for the the inhibit ion of HBsAg production or secretion.

1 7. A compound according to any one of claims I to 10 for the treatment or prophylaxis of HBV infect ion.

18. A compound according to any one of claims 1 to 10, when manufactured according to a process of claim 1 1 .

19. A method for the treatment or prophylaxis of H BV infect ion, which method comprises admin istering an effect ive amount of a compound as defined in any one of claims 1 to 10.

20. The invention as hereinbefore described.